Article pubs.acs.org/JAFC
Curcumin Bioavailability from Enriched Bread: The Effect of Microencapsulated Ingredients Paola Vitaglione,† Roberta Barone Lumaga,† Rosalia Ferracane,† Irena Radetsky,‡ Ilario Mennella,† Rita Schettino,† Saul Koder,§ Eyal Shimoni,‡ and Vincenzo Fogliano*,† †
Department of Food Science, University of Naples, via Università 100, 80055 Portici (NA), Italy Technion Israel Inst Technol, Fac Biotechnol & Food Engn, IL-32000 Haifa, Israel § Karmat Coating Industries Ltd., M.P. Hevel Megido, 19245 Kibbutz Ramot Menashe, Israel ‡
ABSTRACT: Human bioavailability of curcumin from breads enriched with 1 g/portion of free curcumin (FCB), encapsulated curcumin (ECB), or encapsulated curcumin plus other polyphenols (ECBB) was evaluated. Parental and metabolized curcuminoids and phenolic acids were quantified by HPLC/MS/MS in blood, urine, and feces collected over 24 h. The concentrations of serum curcuminoids were always below 4 nmol/L and those of glucuronides 10-fold less. Encapsulation delayed and increased curcuminoid absorption as compared to the free ingredient. Serum and urinary concentrations of ferulic and vanillic acid were between 2- and 1000-fold higher than those of curcuminoids, with ECBB eliciting the highest amounts. Fecal curcuminoids were 6-fold more abundant after ECB than FCB, while phenolic acids after ECBB quadruplicated those after ECB. Curcuminoid encapsulation increased their bioavailability from enriched bread, probably preventing their biotransformation, with combined compounds slightly reducing this effect. Phenolic acids are the major metabolites of curcuminoids and may contribute to their biological properties. KEYWORDS: bioavailability, curcumin, encapsulation, functional food
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INTRODUCTION Curcumin is commonly used in food products, mainly as coloring agent. Several biological properties have been attributed to this compound mainly related to its ability to inhibit NF-kB activation.1 Curcumin has been proposed as a potential therapeutic agent against several non communicable chronic diseases having an inflammatory origin such as neurodegenerative diseases (Alzheimer’s and Parkinson’s disease, multiple sclerosis, epilepsy), cardiovascular diseases (CVD), diabetes, obesity, allergies, and certain types of cancer.2 Although clinical studies in humans proved that curcumin is safe and well tolerated even at very high doses (8−12 g/die), its use as a therapeutic agent is limited by its low bioavailability, poor absorption, rapid metabolism, and systemic clearance.3,4 Drug delivery systems such as nanoparticles, liposomes, microemulsions, and polymeric implantable devices are emerging as viable alternatives that can be used to deliver therapeutic concentrations of various chemopreventive agents such as curcumin, ellagic acid, green tea polyphenols, and resveratrol into the systemic circulation.5 Several absorption enhancers have also been used to improve curcumin bioavailability. Piperine enhanced the bioavailability both in preclinical studies and in studies on human volunteers.6 This was attributed to the ability of piperine in reducing firstpass metabolism.6 Animal studies also demonstrated that inclusion of curcumin into nanoparticles caused at least a 9fold increase in oral bioavailability when compared to curcumin administered alone or with piperine.7 On the other hand, interactions among bioactive compounds that may positively influence oral bioavailability of individual molecules are known for genistein toward epigallocatechingallate8 as well as for several © 2012 American Chemical Society
natural bioactive compounds (quercetin, hesperitin, curcumin, piperin, and naringenin) with P-glycoprotein-inhibiting activity, toward some anticancer drugs.9,10 Encapsulation may confer new properties and potentials to bioactive compounds through modification of physical and nutritional properties.5 This may be of particular interest in the formulation of functional foods, where technological and nutritional aspects must be strictly considered.11 In this respect, selecting suitable coating materials can increase water solubility of bioactive compounds and permit their controlled delivery into the gastrointestinal tract.5 In this framework, the aim of this study was to evaluate the bioavailability of curcumin from different types of bread containing curcumin in different forms: free and microencapsulated in a cellulose derivative coating containing curcumin alone or in combination with a mixture of three bioactive compounds including piperine, quercetin, and genistein. A crossover, randomized, single blind study in healthy subjects was performed. Curcuminoid bioavailability over 24 h following consumption of the breads was assessed by HPLC/MS/MS determining blood, urine, and fecal concentrations of curcuminoids, their metabolites (glucuronides, sulphated, and reduced compounds), and several phenolic acids. Received: Revised: Accepted: Published: 3357
November 5, 2011 January 29, 2012 March 8, 2012 March 8, 2012 dx.doi.org/10.1021/jf204517k | J. Agric. Food Chem. 2012, 60, 3357−3366
Journal of Agricultural and Food Chemistry
Article
Table 1. Composition of the Functional Ingredients Included in the Different Types of Bread core curcuminoids FC (free curcumin) EC (encapsulated curcumin) EC+B (encapsulated curcumin + other polyphenols)
95% 72.68% 66.5%
coating
piperine quercetin
1.0%
1.0%
genistein
cellulose derivative (Ethocell 100)
castor oil
HVO
1.0%
7.48% 6.84%
1.02% 0.93%
15% 13.73%
Figure 1. Study design. Each subject followed this time schedule for each type of curcumin-enriched bread by a crossover double-blind randomized design.
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formulated. Total phenolic acids in these breads were measured. They were at the same amount in all types of bread, and in particular total phenolic acids were 3.7 ± 0.5 mg/100 g of which ferulic acid was 2.2 ± 0.2 mg/100 g, vanillic acid was 1.4 ± 0.5 mg/100 g, and cumaric acid was 0.1 ± 0.05 mg/100 g. In addition, the bread with EC+B also contained 0.01 g of piperine, 0.01 g of quercetin, and 0.01 g of genistein. Depending on the ingredient used, the breads will be hereinafter indicated as: FC bread (FCB), EC bread (ECB), and EC+B bread (ECBB). They were produced in laboratory scale, and curcumin bioavailability upon their consumption was studied. By consuming one portion of bread, subjects ingested 0.8 g (2.1 mmol) of curcumin, 0.2 g (591.7 μmol) of desmethoxycurcumin, and 0.08 g (259.7 μmol) of bisdemethoxycurcumin (3.0 mmol of total curcuminoids). Subjects and Treatment. The protocol of the study was approved by the Ethics Committee of “Federico II” University of Naples (Approval Number: 37/10). Ten healthy subjects, age 31 ± 2 years, BMI of 23.5 ± 1.2 kg/m2, were enrolled. Subjects with gastrointestinal pathologies and/or metabolic disease, those taking anti-inflammatory drugs, or those under controlled diet in the previous 6 months were excluded from the study. Volunteers signed a written informed consent before starting the experimental protocol, consisting of a double-blind randomized crossover study as schematized in Figure 1. Volunteers were asked to follow a polyphenol-free diet for 3 days before and over the experiment days. Thus, they were recommended to exclude from their diet all polyphenol-rich foods and beverages such as fruits, vegetables, chocolate, tea, coffee, wine, beer, supplements, herbal extracts, and whole grains-based foods. Assumption of nonsteroidal anti-inflammatory drugs (NSAID) was also avoided during 1 week before the study. On the experiment day, at 8:00 a.m., 12 h-fasted subjects reached the laboratory and were randomized (using a computer generated random sequence) to receive, in a blinding manner, one of the three types of experimental bread. A different codification of breads by an external researcher allowed the double blindness of the study until the final data analysis. One portion of the bread was served to each participant, and it was entirely consumed at the moment (for breakfast) within 15 min. Before consumption of bread and after 30 min, 1 h, 2 h, 4 h, and 6 h, blood drawings were performed. Urine volume was measured over 24 h, and 10 mL samples were collected before and at 0−2 h, 2− 4 h, 4−6 h, 6−8 h, 8−10 h, and 10−24 h time intervals postbread ingestion. After 6 h from breakfast ingestion, subjects were given another portion of the same bread type they had consumed in the morning, then they left the research center and consumed their lunch (always choosing among allowed foods). The second bread portion
MATERIALS AND METHODS
Chemicals. All chemicals and reagents were of analytical grade. Methanol, water, and acetonitrile were from Merck (Darmstadt, Germany); ethyl acetate, glacial acetic acid, and hydrochloric acid were from Clean Consult International (Lodi, Italy); formic acid (98% purity) and butylated hydroxytoluene (≥99%) were obtained from Sigma (St. Louis, MO). All analytical standards chlorogenic acid (95%), ferulic acid (99%), 4-hydroxyphenylacetic acid (HPA, 98%), 3-(4-hydroxyphenyl)propionic acid (HPP, 98%), vanillic acid (97%), and curcumin (≥80%) were purchased from Sigma (St. Louis, MO). Curcumin Ingredients and Breads. Three types of curcumin containing ingredients were used, free curcumin (FC), encapsulated curcumin (EC), and encapsulated curcumin, plus three bioactive compounds, piperine, quercetin, and genistein (EC+B). FC was a 95% pure curcuminoid extract from turmeric and was constituted by 79% curcumin, 19% desmethoxycurcumin, and 2% bisdesmethoxycurcumin. EC and EC+B were obtained by fluidized bed spray coating, followed by bottom spray. Curcumin was encapsulated by double coating, whereas the inner coating material of microcapsules was constituted by cellulose derivative (Ethocel 100, Dow Chemicals) as a first layer, and hydrogenated vegetable oil (HVO) as an external layer. Ethocel 100 (88% Ethocel 100 and 12% liquid castor oil, as emulsifier) was dissolved in 80% acetone and 20% methanol to get a 4% w/w solution for coating, and hydrogenated vegetable oil was melted by heating to 95 °C prior to coating. The particles of curcumin were placed at the bottom of the chamber and blown upward by hot air. The coating polymer solution (Ethocel 100) and HVO were sprayed upward in the same direction, one by one. In this way, curcumin particles pass through a simultaneous coating (drying) environment upward by reaching the top of the chamber; the partially coated particles move downward and undergo further drying until the desired coat thickness is reached. Finally, EC and EC+B contained 72.7% and 66.5% of curcuminoids, respectively; in EC+B, piperine, quercetin, and genistein, 1.0% of each, were also present (Table 1). All combined compounds were encapsulated one by one with a single layer of polymer solution (Ethocel 100) by different amounts of coatings (0−20% coat), mixed together to achieve controlled release mechanism. Final moisture, for both EC and EC+B, was
